Cytolytic peptides or cytolysins have previously been used to release active agents or “payload” from liposomes or cells. The mode of action for such peptides involves perturbation of the liposome or cellular membrane. These peptides include toxins from insects, fish, antibiotic peptides and synthetic peptides such as melittin, alamethicin, gramicidin, magainin and pardaxin, GALA, KALA, hemagglutinin subunit HA-2. Natural potent cytolytic peptides are found widely from insects to mammals, particularly as antimicrobial peptides or defensins, where they are involved in innate defence at mucosal membranes and as cytolysins in lymphocytes. In order to target and localise the cytolytic action of such peptides, a number of specific steps e.g. activating synthesis, release from lysosomes, cleavage of pro-peptides is required. The biological delivery activity of such peptides is tightly controlled at the cellular and molecular levels. Biologically, cytolysin activity is cloaked by sophisticated mechanisms available within and between cells butthese mechanisms are diagnostically and therapeutically less exploitable. This has therefore hindered the use of cytolysins in diagnostic and therapeutic applications.
Whilst much sought after, there are remarkably few simple and rapid homogeneous biodetection methods.
Owing to their inferior sensitivity and non specific variable background, compared to the automated heterogeneous technology which is now widespread in immunodiagnostics and high throughput screening, it has not always been possible to develop homogeneous assays for different analytes. Liposomes have, previously, been utilised in homogenous assays using complement-mediated lysis (Anal. Biochem. 118 (1981) 286-293.) However, such assays are considered unreliable as they involve many labile components, any one of which may become inactivated eliminating payload release. The development of a homogenous liposomal assay using non-specifically labelled digoxin melittin as the lytic agent was reported as an alternative to the complement assay (J.Immunol. Methods. 70 (1984)133-140). This method, however, has not gained widespread use as the preservation and stability of lytic activity, as well as solubility of the conjugates posed problems largely restricting the use of this cytolytic peptide to measure digoxin. This may be expected, primarily due to the uncertainties involved in the production of useful cytolysin conjugates by relying solely on natural peptides with multiple labelling sites, most of which are critical for peptide function and, thus, not ideally placed for retaining high activity if modified. Furthermore, the degree of modulation in activity of these conjugates is often inadequate resulting in high background signals. Owing to these difficulties when using natural peptides, others have used conjugates with a larger cytolysin, namely phospholipase C, non-specifically labelled with analytes (J.Immunol. Methods 170 (1994) 225-231). Such conjugates had superior solubility and greater retention of activity after modification. However, only 75 to 85% activity was specifically inhibited in the presence of anti-serum, which is comparable to the level of inhibition normally used for measuring digoxin with melittin-oubain conjugates. A reliable assay should only permit the release of marker molecules upon external trigger and the background leakage should approach zero or at least remain constant over the assay period. To our knowledge neither of these conditions have been satisfactorily addressed by homogeneous liposomal assays, without changing to a heterogeneous assay configuration. Consequently, with such assays there is always a danger of the background signal progressively interfering with the analyte dependent signal. Some of the long term background problems arising from the use of liposome reagents per se can be overcome by the use time resolved fluorimetry, in which a larger molecular weight protein chelator conjugate is encapsulated in the liposomes, allowing fluorescent detection upon cytolysin mediated complexing with ions such as Eu3+(Anal. Biochem 238 (1996) 208-211). Even with these assays, the inherent problems of the non-specific lysis by uninhibited conjugates as well as optimising conjugates to produce adequate activity, remain. Consequently, such assays need to be performed under well-controlled laboratory conditions and at fixed times against the varying background signal.
Liposomes have been used more widely in drug delivery rather than in diagnostic applications and or as imaging agents, however, in all cases there has been little progress made with the use of liposomes, efforts being mainly devoted to developing different lipid formulations to try to achieve controlled and quantitative release of active agent or payload in response to a trigger.
For a reliable assay, the release of detectable marker molecules should only occur in response to an external trigger and any leakage of marker molecules should be minimal for example, approaching zero, or at least remain constant over the assay period. Consequently, in such assays there is always a danger of background signal or interference caused by the progressive release of marker molecules.
Our earlier patent application WO98/41535 (PCT/GB98/00799) describes peptides which can be efficiently cloaked and used to release a “payload” in a controlled manner. The peptides disclosed in that application were non-responsive to pH change particularly over a narrow range between pH 6.5 and 7.4. On the contrary, in most cases, lowering of the pH would result in the lowering of peptide activity. A number of pH sensitive peptides have been used to release payload from liposomes under acidic conditions (Advanced Drug Delivery Reviews 38 (1999) 279-289). For these peptides the triggering range is, however, far from physiological pH, usually requiring pH values lower than 6 to release payload from liposomes.
GALA is one of the most efficient pH specific peptide. For this peptide Calcein release from liposomes has been demonstrated at values lower than 6. There are many other pH specific peptides, such as Influenza virus HA-2 N-terminal peptide, EALA, JTS1 and Rhinovirus VP-1 N-terminal peptide which have been shown to release liposome contents in low pH environments such as the endosome where the pH is reported to be well below 6 and typically 5. There are several pH sensitive peptides known in the literature to destabilize liposome membranes. However they are usually triggered at very low pH (5.5) and consequently have found little or no use in drug delivery, for example, to tissues or tumours where the pH difference between normal and diseased areas can be less than a one pH unit. Their major use thus remains endosome delivery.
The strategy of micro-environmental pH change in tissues to induce preferential release of drugs from liposomes requires peptides to respond over a narrow pH change, closer to the physiological range. To our knowledge there are no reported peptides which trigger release of payload from liposomes efficiently and close to physiological pH levels of 7.4 while their background biological activity remains low or zero at or close to pH 7.4.
A peptide named “helical erythrocyte lysing peptide” (HELP) (Protein Eng. (1992), 5, 321) is known to lyse red blood cells and has been shown to trigger release of haemoglobin below pH 6.5 only. This peptide is, however, specific to lysing cells and there are no reports showing lysing of liposomes. We have shown that liposomes could not be lysed in a pH specific manner using this peptide.
WO97/38010 relates to fusogenic liposomes and delivery systems for transporting-materials such as drugs, nucleic acids and proteins. These systems work by fusion of liposomes and at pH values lower than 6.